8/4 Colony PCR of putative mtrB samples

We picked 24 colonies each of old putative P98 and newly transformed putative P98+63 for colony PCR using the BioBrick primers and 55°C annealing for 30 cycles. If the colonies contained mtrB, a band at ~2.1 kb should appear. No colonies appeared to contain mtrB.

Ligations and transformations 08/06

We ligated P105 and P1/P3 not BB, and mtrB not BB and P1/P3 not BB. We tried letting the ligation reactions run for 5 and 10 minutes. We used P1/P3 not BB from 08/05 and from another date (older). We transformed using TOP10 and DH5α. We also retransformed P108, the Lac QPI with a high constitutive promoter in a p15A vector.

TOPO cloning

08/04

P1, mtrB

These had a few white colonies. Colony PCR, however, indicated that the correct PCR product was not incorporated.

ompR

All colonies were blue.

HO-pcyA

No colonies.

Transforming in XL10-Gold cells

Plate

Marker

# Colonies

Description

XL10-Gold P75a + P63b (1:1)

Kan

2

No fluor

XL10-Gold P75b + P63b (1:1)

Kan

6

1 fluor

XL10-Gold P75a + P63b (6:2)

Kan

0

XL10-Gold P75b + P63b (6:2)

Kan

1

fluor

XL10-Gold P75a + P63b (7:1)

Kan

1

no fluor

XL10-Gold P75b + P63b (7:1)

Kan

2

both fluor

XL10-Gold Dephos P75a

Kan

1

XL10-Gold Dephos p75b

Kan

0

XL10-Gold P3 + (P39+p51) 2/6

Kan

XL10-Gold P3 Topo w/ XGAL

Amp

XL10-Gold P1 Topo w/ XGAL

Amp

XL10-Gold mtrB Topo w/ XGAL

Amp

XL10-Gold pUC18

Amp

100s

White, very small

XL10-Gold negative control

Amp

many

extremely small

XL10-Gold negative control

Kan

0

Ligations

HO-pcyA, ompR, CDF

All three ligations yielded >50 colonies on the TOP10 cell plate with a 5 minute ligation in all combinations (HO-pcyA and ompR with P1 and old P3; CDF with P3). The P90 (CDF+P3) did not fluoresce. The DH5α cells did not have nearly as many (<5) and ligation times (10min, 30 min) did not appear to be significant.

Colony PCR

We picked at least 18 colonies from each ligation set, patched a master plate, inoculated liquid cultures, and performed 12.5 μL colony PCR reactions. The BBp/sfx primer pair was used, annealing temperature was 50°C for first 10 cycles and 54°C for rest of the cycles. 2 controls were performed: one using a picked P20 colony, and another with miniprepped P20 plasmid.

Digests, 1-8

We ran the digests to see if PCR mutations could have introduced an internal cut site. However, even with the small amount of purified digest we had left, we could see a properly sized band for each of P1, ompR, and HO-pcyA PCR products.

1-16

4, 6, 14 look like they have potentially correct products, so liquid cultures were grown up.

17-24: P105+P1 ligation colony PCR (2.5kb)

None appear to be correct.

25-48: mtrB+P63 ligation colony PCR (2.2kb)

27, 28 picked.

RE digests 08/09

Lane 1: 1 kb ladder

Lane 2: mtrB+63 #28 cut XP (~2200+3.1kb)

Lane 3: E1P3A-P38+51C cut SP (4155)

Lane 4: E1P3A-P38+51D cut SP (4155)

Lane 5: E1P3A-P38+51B cut SP (4155)

49-64: mtrB+P97 ligation colony PCR (2.1kb)

51 picked.

65-80: mtrB'+P3 ligation colony PCR (2.3kb)

65, 69, 70, 78, 79, 80 picked.

81-96: mtrB'+P1 ligation colony PCR (2.3kb)

82, 87, 95 picked.

RE digests 08/07

Lane 1: 1 kb ladder

Lane 2: E1P1+P17A cut EX (3652)

Lane 3: E1P1+P17C cut EX (3652)

Lane 4: E1P1+P17D cut EX (3652)

Lane 5: E2P1+P17A cut EX (3652)

Lane 6: E2P1+P17B cut EX (3652)

Lane 7: P108 cut SP (4155)

Lane 8: P45 cut XP (876; 2079)

Ligations 08/08

We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 2:6. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

Transformation results 08/09

Plasmid

Amount of DNA

Strain

Ligation ratio (vector:insert by volume)

Number of colonies

P108 (vector control)

7 ul

100 ul DH5-alpha

2 (vector)!6 (water)

0

P45+108

7 ul

100 ul DH5-alpha

2:6

1

P17 in p15A (vector control)

7 ul

100 ul DH5-alpha

1 (vector):7 (water)

188

(P17 in p15A)+P39

7 ul

50 ul TOP10

1:7

1

(P17 in p15A)+P39

7 ul

100 ul DH5-alpha

1:7

0

(P17 in p15A)+P38

7 ul

50 ul TOP10

1:7

3

(P17 in p15A)+P38

7 ul

100 ul DH5-alpha

1:7

0

Colony PCR 8/9

We picked all of the noncontrol colonies and one control colony for colony PCR using the BBp/sfx primers.

1.2% E-gel visualized using EtBr/UV

Lane

Contents (approx. expected band size)

1

P39+P17 in P1/3- col#1 (~940bp)

2-5

P38+P17 in P1/3- col#2-5 (~940bp)

6

1kb ladder

8-9

P108+P45 (same sample) (~2.3kb)

10

ligation control (no insert)

Since the bands are at least plausible, the P38/39 in P1/3 samples were grown up and miniprepped.

Retransformation 8/9

We retransformed 5μL of the P39+17 in P1/3 ligation into 50μL DH5α and obtained two colonies.

Colony PCR shows that one of these does not contain the right construct. The other was set up in 5ml culture.

New ligations and transformations 08/10

We retransformed our old P108+45 (2:6 vector to insert) ligation and the old vector control. We also did this ligation over with a 2:1 molecular ratio of insert to vector. Both P108 (4155 bp) and P45 (876 bp) were about 17 ng/μL. We used a ratio of 5.4:2.3 vector to insert by volume.

Transforming LacZ parts

We attempted to transform the following parts: I732017, E0435, E0435, I732005 into our competent DH5α. E0435 (on CM) had 0 colonies). The others (on Carb) had 10-20 extremely small colonies (after 16+hrs of incubation) each. We picked 2 of each for liquid culture, but none grew.